Molecular Screening of Rice Blast Resistance

The PCR was performed using a total volume of 10 µl reaction mixture, containing 3 µl of 30 ng of template DNA and 0.5 µl of 5 Pico moles of each primer (Invitrogen), 1 µl of reaction buffer, 1 µl of dNTPs and 0.2 µl of Taq DNA polymerase. The PCR profile used was one cycle of initial denaturation at 95 °C for 5 min, followed by 35 cycles of amplification (each cycle of 1 min denaturation at 95 °C, 30 secs annealing at 55 °C, 30 secs extension at 72 °C), followed by a final extension of 72 °C for 7 min and stored at 4 °C. The amplified products were resolved in 3.5% Agarose gel with 0.5 X TBE buffer containing 0.1 μg/ml of Syber safe (S33102, Invitrogen) and documented in the Gel documentation system^{21 (link)}. Gel scoring was carried out based on the donor and recurrent parent banding patterns and their corresponding product sizes. The allele corresponding with Matatag 1 was considered resistant allele. Similarly, allele matched with recipient parent BRRI dhan71 scored as a susceptible allele. Whereas if both the alleles (susceptible and resistant) were observed in one sample, they were considered heterozygotes.